Viral-mediated bacterial mortality and the prevalence of lysogeny are two key parameters for understanding the role of viral activity in aquatic ecosystems. The viral production assay is most commonly used to assess these parameters, with lytic and mitomycin C-induced viral production rates prevalently extracted using the linear regression or increment-based (VIPCAL) approach. A literature survey shows that 64% of the 89 viral production studies used the linear regression approach for lytic and 48% employed VIPCAL for lysogenic viral production rates. Our comparative evaluation highlights significant differences between these two approaches of estimating viral production rates. To refine estimations, we enhanced VIPCAL to VIPCAL-SE by incorporating standard error of the means to rigorously identify maxima–minima pairs, accounting for biological and ecological variabilities between replicates. We also included a bacterial net generation time endpoint to reduce estimation bias due to potential secondary infections, particularly relevant in more productive ecosystems. VIPCAL-SE is now available as a part of the viralprod R package and provides an opportunity for further standardisation in the field of aquatic viral ecology.

With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.

Chromatin is intrinsically repressive, limiting access to DNA, implying a major regulatory role. Studies with nuclei support this model. However, we have shown previously that genomic DNA is almost completely accessible in living budding yeast and human cells, except for centromeric chromatin. The fission yeast, Schizosaccharomyces pombe, possesses heterochromatin similar to mammalian heterochromatin at the pericentromeric repeats, telomeres and the silenced mating type loci. S. pombe heterochromatin is marked by histone H3K9 di- and tri-methylation (H3K9me2/3) and heterochromatin protein 1 (HP1/Swi6), potentially repressing genes by preventing access to the DNA. Here, we developed a copper-inducible DNA methyltransferase system to measure accessibility in living S. pombe cells. We find that euchromatin and heterochromatin are generally accessible, indicating that heterochromatin does not represent a significant block to DNA methyltransferases in vivo. S. pombe centromeres are much more accessible than budding yeast and human centromeres. In contrast, S. pombe chromatin is mostly inaccessible in isolated nuclei, primarily due to tight nucleosome spacing on gene bodies, with very little linker DNA. We conclude that S. pombe euchromatin and heterochromatin are both highly dynamic in vivo, suggesting that the H3K9me/HP1 system does not repress transcription by preventing access to DNA.

Creating artificial organelles that sequester and release specific proteins in response to a small molecule in mammalian cells is an attractive approach for regulating protein function. In this work, by combining phase-separated condensates formed by the tandem fusion of two oligomeric proteins with a trimethoprim (TMP)-responsive nanobody switch for GFP (GFPLAMA; ligand-modulated antibody fragment), we developed a synthetic condensate system that initially sequesters GFP-tagged proteins within condensates and rapidly releases them into the cytoplasm upon TMP treatment. The released proteins can then be resequestered by washing out the TMP. This system enabled user-defined, temporal, rapid, and reversible control of cellular processes, including membrane ruffling mediated by exogenously expressed GFP-Vav2 and modulation of the cellular localization of endogenous ERK2-GFP generated by genome knock-in. Our results highlight the utility of the GFPLAMA-based synthetic condensate platform as a novel, chemically switchable tool for regulating protein function through controlled protein sequestration and release in mammalian cells.

Bacteria and archaea encode on average ten antiphage systems. Quorum sensing, cellular, or transcription factors can regulate specific systems (CRISPR-Cas, CBASS). Yet, a systematic assessment of antiphage systems expression patterns is lacking. Here, we combine publicly available RNA-seq data from 14 different species with an original RNA-seq dataset of 15 Escherichia coli strains across six environmental conditions and two growth stages. Using this data, we explore the transcription patterns of 236 antiphage systems from 81 types. Defense system expression is variable along environmental, physiological, as well as spatial gradients, and can correlate with cellular physiology and mobile genetic element activity. We identify antiphage systems as cohesive but complex transcriptional units, find coordinated expression of defense islands possibly underpinned by local regulators, and demonstrate the functional relevance of differential expression in native systems. Together, these results suggest that environmental and physiological factors regulate prokaryotic immunity and may prime bacteria for infection.

Xyloglucan (an alpha-1,6-xylosyl-substituted beta-1,4-glucan) is a major hemicellulose of the primary cell wall of many plants and an important growth substrate for biomass-degrading bacteria in diverse ecological niches, including the gut microbiome and hot springs. In Gram-positive bacteria, xyloglucan is deconstructed into soluble oligosaccharides in the extracytoplasmic space before import by ATP-Binding Cassette (ABC) transporters, but the structural basis for this process remains poorly understood. Here, we identified an ABC transporter for xyloglucan uptake (Athe_2052-2054) in the Gram-positive, plant biomass-degrading thermophile Anaerocellum bescii, which is conserved across the Anaerocellum genus. We solved the apo crystal structure of its extracellular substrate-binding protein (SBP), Athe_2052, revealing a unique tertiary fold found only in a small subset of SBPs that bind complex oligosaccharides. This structure represents the first ABC SBP known to bind xyloglucan oligosaccharides. Biophysical analysis showed that while Athe_2052 binds unsubstituted beta-glucan chains, recognition of xyloglucan side chains in the binding pocket markedly increases affinity (Kd = 14 nM) for xyloglucan heptasaccharide (XXXG), the principal oligosaccharide released during xyloglucan deconstruction. Molecular modeling revealed that xyloglucan heptasaccharide, owing to its branched substitutions, is bound in a distinct conformation compared to unsubstituted beta-glucans. This represents a unique mode of xyloglucan recognition driven by alpha-linked side-chain interactions rather than beta-glucan backbone recognition alone. Together, these findings provide the first structural basis for xyloglucan oligosaccharide recognition by an ABC transporter in Gram-positive bacteria.

Pseudomonas aeruginosa is a human opportunistic pathogen, capable of producing a wide range of metabolites, including pyomelanin. This pigment results from alterations in tyrosine catabolism. Melanin synthesis from tryptophan has never been reported in Pseudomonas. In this study, we describe a tryptophan-derived melanin in P. aeruginosa PAH, a strain that was isolated from a fibrocystic patient. PAH produced a brown pigment when grown in LB or L-tryptophan-supplemented media. Structural analysis revealed this pigment was composed by two fractions differing in NaOH solubility: a soluble one consistent of pyomelanin, and an insoluble fraction with a complex structure containing substituted indolic units. A pyomelanin inhibitor enhanced total melanin synthesis, mainly the insoluble fraction, and a tryptophan 2,3-dioxygenase inhibitor decreased pigment formation. Metabolomic profiling identified distinct indolic compounds and low levels of anthranilate in PAH cultures. Genomic and transcriptomic analyses revealed the presence of mutations and downregulation of genes related to pyoverdine biosynthesis. Furthermore, iron supplementation in the culture medium reduced melanin production. Overall, tryptophan arises as a key compound for melanin production in PAH, expanding the diversity of melanins synthesized by this genus. Furthermore, iron deprivation emerges as a critical factor triggering melanin biosynthesis, probably as a survival strategy enabling persistence in the fibrocystic lung environment.

Several luciferases have been developed for imaging and biosensing, and the collection continues to grow as new applications are pursued. The current workflow for luciferase optimization, while successful, remains laborious and inefficient. Mutant libraries are generated in vitro and screened, “winning” mutants are picked by hand, and the isolated sequences are subjected to additional rounds of mutagenesis and screening. Here, we present a streamlined platform for luciferase engineering that removes the need for manual library generation during each cycle. We purposed an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of a well-known luciferase (GeNL). Short cycles of culturing and screening were sufficient to evolve the enzyme, with no repetitive manual library generation necessary. New GeNL variants were identified that exhibit improved light outputs with a noncognate and inexpensive luciferin. We further characterized the novel luciferases in cell models. Collectively this work establishes OrthoRep and continuous hypermutation as a viable method to engineer luciferases, and sets the stage for more rapid development of bioluminescent reporters.

Metabolic regulation─the dynamic biochemical network governing energy transduction, substrate conversion, and metabolic flux─represents a fundamental determinant of microbial viability and functional output. While temporal metabolite fluctuations provide critical insights into metabolic network dynamics, conventional analytical platforms face fundamental limitations in real-time monitoring within native microbial environments. This study presents a novel whole-cell electrochemical biosensing platform integrating carbon dot (CD)-engineered Escherichia coli with advanced cyclic voltammetry (CV) for dynamic metabolic interrogation. This biohybrid system synergizes microbial biochemical specificity with CD-enhanced electron transfer efficiency, achieving nearly a 20-fold amplification in the electrochemical signal amplitude through quantum-enhanced charge transport mechanisms. The platform enables precise quantification of redox-active metabolites via distinct voltammetric fingerprints, as demonstrated through the detection of lactic acid─a pivotal biomarker in industrial biotechnology and clinical diagnostics. Featuring a modular bioarchitectural design, this technology permits seamless adaptation across diverse microbial systems, offering unprecedented capabilities for real-time bioprocess optimization, dynamic metabolic pathway analysis, and pathogen metabolic profiling. By interfacing nanomaterial-enhanced electrochemistry with synthetic biology, our platform surmounts traditional analytical constraints, establishing a versatile analytical tool for spatiotemporal mapping of metabolic networks in complex biological matrices.

Phylogenetic trees play a fundamental role in elucidating evolutionary relationships among taxa. Clustering taxa remains a major challenge across diverse biological domains such as cancer genomics, microbial systematics, and phylogenomics. Several methods partition taxa in phylogenetic trees into clusters, but these approaches face key limitations. Many rely on arbitrary distance thresholds that are contingent on a-priori information. These thresholds can be hard to ascertain, are subject to bias, and may vary across studies—hindering meaningful interpretation and comparison. More broadly, many methods lack rigorous definitions of what constitutes a cluster, and depend on heuristics that restrict the cluster search space since enumerating all possible clusters is computationally infeasible for large trees. Here, we present PhytClust, a threshold-free algorithm that partitions taxa in phylogenetic trees into monophyletic clusters. PhytClust provides an exact solution to the problem of finding an optimal set of clusters in a phylogenetic tree that minimizes the total intra-cluster branch lengths for a given number of clusters. It then determines the optimal number of clusters using a validity index. PhytClust yields an exact and efficient clustering solution that reflects the tree's topology and genetic distances. In simulated datasets, PhytClust outperforms existing methods in both speed and accuracy and scales to trees with more than a hundred thousand taxa. We apply PhytClust across cancer genomics, avian phylogenomics, bacterial and archaea phylogenetics, and plant genomics to demonstrate PhytClust's varied applicability. By providing a standardized method for taxa clustering within phylogenetic trees, PhytClust yields reproducible, optimal and computationally efficient clusters.

Collective behavior is a defining property of multicellular systems, where coordinated outcomes emerge from local cell-cell interactions. Yet the quantitative rules linking single-cell decision-making to tissue-scale organization remain poorly resolved. Here, we develop a quantitative framework that defines an order parameter predicting when initially disordered colonies undergo a transition to ordered fate alignment and when minimal, localized inputs can redirect their collective state. This analysis reveals a distinct control regime in which multicellular assemblies become susceptible to a single engineered guide cell. We validate these predictions by introducing guide cells that integrate into unperturbed colonies and redirect fate patterns within the theoretically defined control windows. Together, these results connect single-cell decision rules to emergent tissue-level organization and establish a generalizable biological control strategy in which a minority engineered subset can reliably redirect the developmental trajectory of a much larger multicellular population.